NMR Spectrometry

Researchers in the UW department
of chemistry have developed powerful probes of molecular
structure using the technique of nuclear magnetic resonance
spectrometry (NMR) to tackle, among other tasks, the problem of
determining the structure of large, complex biomolecules like
DNA. Moreover, UW faculty have teamed up with scientists at
Pacific Northwest National Laboratory (PNNL) in Richland,
Washington, to design and construct the world's most powerful
NMR instrument.

The NMR technique helps scientists deduce the arrangement of
atoms within a molecule--its molecular structure.
Molecules consist of atoms linked together by chemical bonds.
Atoms, in turn, consist of a dense, compact core, called the
nucleus, surrounded by what could be likened to a "cloud" of
electrons. The electrons participate in chemical bonding,
providing the chemical "glue" that holds atoms together to make
a molecule. The exact arrangement of the atoms determines the
identity of the molecule, its structure, and its chemical
characteristics. But since atoms and molecules are too small to
see, chemists have devised a number of complementary strategies
to deduce how atoms are linked together.

One such strategy is NMR. The method is based on measuring
the absorption of very-low-energy radio waves by the nuclei of
atoms in a molecule which has been placed in a magnetic field.
The particular distribution of electrons around a nucleus
influences the precise frequency of the radio waves that are
absorbed. That electronic distribution in turn is a function of
the chemical bonds and neighboring atoms surrounding the
nucleus. So, by measuring the frequencies at which atoms absorb
energy, scientists may gain clues about the local environment
of each atom and can then deduce the overall structure of the
molecule. In practice, several different but related NMR
procedures are often used in concert to piece together a
picture of a complex structure. An extension of nuclear
magnetic resonance spectrometry to the imaging of biological
tissues is magnetic resonance imaging, or "MRI."

In about 1980, the UW chemistry department under the
direction of then-chairman Alvin L. Kwiram began an initiative
to build up its program of NMR research. Kwiram recruited Brian
Reid, and subsequently Gary Drobny, to the chemistry faculty to
launch a new era in NMR research. Reid, a biochemist, began
working on the structure of DNA in solution, using
sophisticated, high-resolution NMR techniques devised and
refined by Drobny, a physical chemist.

Prior to this work, the only way to determine the structure
of such a molecule was to grow a solid crystal of it, and then
analyze it by x-ray crystallography; but crystallizing the
substance, even if possible, can cause structural distortions
compared to the configuration of the molecule in its natural
state in biological fluids.

Reid and colleagues pioneered a groundbreaking approach to
solve the structure of DNA in solution by measuring literally
hundreds of inter-nuclear distances using NMR and then
developing a computer algorithm to deduce the molecular
arrangement of the atoms. Thus, for the first time, a
revolutionary new method was available for determining the
structure of large molecules in solution to complement the
century-old traditional method of x-ray crystallography with
crystalline materials. These NMR methods are now among the most
powerful tools used by chemists and life scientists to study
the solution structure of biomolecules.

More recently, Reid has used a similar procedure to
determine the structure of hybrid double
helices--biomolecules similar to double stranded, helical
DNA but containing one strand of ribonucleic acid (RNA) and one
strand of deoxyribonucleic acid (DNA). "These hybrids are very
important in such processes as the life cycle of retroviruses
like HIV [Human Immunodeficiency Virus]," the cause of AIDS,
explains Reid.

Reid and colleagues have established for the first time the
unique structure of the hybrid double helix. "It is distinctly
different from the DNA double helix or RNA double helix," says
Reid. "It's the uniqueness of the hybrid that explains how the
enzyme ribonuclease-H works--an enzyme that is very
important in the HIV replication cycle, and on which an awful
lot of research on AIDS is being focused."

In addition to the hybrid double helix work, Reid and his
students have been using NMR to study abnormal base pairing in
DNA. Although Watson and Crick established that in the DNA
double helix, the base adenine (abbreviated "A") pairs with
thymine (T), and guanine (G) pairs with cytosine (C), Reid's
group has established that, in certain sequences, G can pair
with A and vice versa. In one of these sequences, which is
repeated thousands of times and makes up 5-6% of human DNA, the
DNA strand pairs with itself instead of with its complementary
strand, using GA pairing instead of GC pairing to form what
Reid calls "sticky DNA." He feels that this stickiness may
explain how duplicated chromosomes are segregated equally into
daughter cells at mitosis. "Although the process is poorly
understood at the molecular level, the accurate segregation of
duplicated chromosomes during cell division is the basis
underlying all of genetics and evolution" says Reid.

An unexpected spin-off from Reid's exploration of GA base
pairing in DNA is already shedding some light on a newly
discovered class of inherited genetic diseases known as
triplet-repeat diseases. These include Huntington's disease and
a variety of neuromuscular dystrophies and atrophies; recently
they have all been shown to involve the expansion of a string
of adjacent GCA triplets in the corresponding target genes
(e.g., GCA GCA GCA GCA GCA, etc.). The expansions range from
10-20 repeats in the normal population to hundreds of repeats
in those with the disease.

These diseases all show the phenomenon of anticipation—that
is, increasingly longer repeats within the affected gene from
one generation to the next, resulting in increasingly severe
symptoms and earlier age of onset. Also, the diseases exhibit
autosomal dominance, in which only one of the two chromosomes
need be affected to cause the disease. "It's chemically
fascinating that in all these diseases it is always the GCA
word (out of the 64 words in the genetic dictionary) that
expands," says Reid. He suspects that his unusual GA pairing is
involved in the expansion process.

Meanwhile, UW chemistry professor Gary Drobny and colleagues
have been working on the design and construction of the world's
first 1-Gigahertz NMR spectrometer in collaboration with Paul
D. Ellis of the Environmental and Molecular Sciences Laboratory
(EMSL) at PNNL in Richland, Washington, operated by Battelle
for the U. S. Department of Energy. The 1-Gigahertz
spectrometer will be one of the centerpieces in an array of
advanced instruments and computing tools for the study of
environmental systems at the new EMSL facility.

One Gigahertz (GHz), equivalent to 1,000 Megahertz (MHz) or
10 Hertz (cycles per second), is
an indirect measure of the strength of the magnet used in the
instrument. (It reflects the resonance frequency for protons in
that field strength). The new magnet, which is being
constructed for the project by Oxford Instruments in the U.K.,
will be one of the largest magnets of its type ever made, says
Drobny. It will operate at a temperature of 1.8 Kelvin and will
employ special superconducting alloys such as niobium-tin and
niobium-titanium in order to achieve its powerful field.

The quest for ever higher field strength is important
because in large molecules, NMR lines overlap, which limits the
ability to resolve them and obtain structural information. But
the amount of overlap decreases as the strength of the magnetic
field increases. Until this project was launched, most
researchers had access, at best, to instruments with field
strengths of 500 MHz or lower.

The new apparatus represents a significant increase in
resolving power, permitting the study of larger molecules than
ever before, as well as of solids, especially biomaterials and
catalysts. The spectrometer will be available part of the time
for use by scientists elsewhere, serving as a "prime resource"
for efforts nationwide in environmental and biological
sciences, says Ellis.

It should be noted that none of these developments in
super-high field NMR would have been possible in the Pacific
Northwest without the strong support of the Murdock Charitable
Trust.